Angiogenesis, by definition, is the formation of new capillaries and blood vessels within living tissues; and is a complex process first recognized in studies of wound healing and then within investigations of experimental tumors. Angiogenesis is thus a dynamic process which involves extracellular matrix remodeling, endothelial cell migration and proliferation, and functional maturation of endothelial cells into mature blood vessels [Brier, G. and K. Alitalo, Trends Cell Biology 6: 454–456 (1996)]. Clearly, in normal living subjects, the process of angiogenesis is a normal host response to injury, and as such, is an integral part of the host body's homeostatic mechanisms.
It will be noted and appreciated, however, that whereas angiogenesis represents an important component part of tissue response to ischemia, or tissue wounding, or tumor-initiated neovascularization, relatively little new blood vessel formation or growth takes place in most living tissues and organs in mature adults (such as the myocardium of the living heart) [Folkman, J. and Y. Shing, J. Biol. Chem. 267: 10931–10934 (1992); Folkman, J., Nat. Med. 1: 27–31 (1995); Ware, J. A. and M. Simons, Nature Med. 3: 158–164 (1997)]. Moreover, although regulation of an angiogenetic response in-vivo is a critical part of normal and pathological homeostasis, little is presently known about the control mechanisms for this process. A number of different growth factors and growth factor receptors have been found to be involved in the process of stimulation and maintenance of angiogenetic responses. In addition, a number of extracellular matrix components and cell membrane-associated proteins are thought to be involved in the control mechanisms of angiogenesis. Such proteins include SPARC [Sage et al., J. Cell Biol. 109: 341–356 (1989); Motamed, K. and E. H. Sage, Kidney Int. 51: 1383–1387 (1997)]; thrombospondin 1 and 2 respectively [Folkman, J., Nat. Med. 1: 27–31 (1995); Kyriakides et al., J. Cell Biol. 140: 419–430 (1998)]; and integrins αvβ5 and αvβ3 [Brooks et al., Science 264: 569–571 (1994); Friedlander et al., Science 270: 1500–1502 (1995)]. However, it is now recognized that a major role is played by heparan-binding growth factors such as basic fibrocyte growth factor (bFGF) and vascular endothelial growth factor (VEGF); and thus the means for potential regulation of angiogenesis involves the extracellular heparan sulfate matrix on the surface of endothelial cells.
Research investigations have shown that heparan sulfate core proteins or proteoglycans mediate both heparin-binding growth factor/receptor interaction at the cell surface; and that accumulation and storage of such growth factors within the extracellular matrix proper occurs [Vlodavsky et al., Clin. Exp. Metastasis 10: 65 (1992); Olwin, B. B. and A. Rapraeger, J. Cell Biol. 118: 631–639 (1992); Rapraeger, A. C., Curr. Opin. Cell Biol. 5: 844–853 (1993)]. The presence of heparin or heparan sulfate is required for bFGF-dependent activation of cell growth in-vitro [Yayon et al., Cell 64: 841–848 (1991); Rapraeger et al., Science 252: 1705–1708 (1991)]; and the removal of heparan sulfate chains from the cell surface and extracellular matrix by enzymatic digestion greatly impairs bFGF activity and inhibits neovascularization in-vivo [Sasisekharan et al., Proc. Natl. Acad. Sci. USA 91: 1524–1528 (1994)]. Ample scientific evidence now exists which demonstrates that any alteration of heparan sulfate (HS) chain composition on the cell surface or within the extracellular matrix which is initiated by means of an altered synthesis, or a degradation, or a substantive modification of glycosaminoglycan (GAG) chains can meaningful affect the intracellular signaling cascade initiated by the growth factor. The importance of heparan sulfate in growth factor-dependent signaling has become well recognized and established in this field.
Heparan sulfate (HS) chains on the cell surface and within the extracellular matrix are present via binding to a specific category of proteins commonly referred to as “proteoglycans”. This category is constituted of several classes of core proteins, each of which serve as acceptors for a different type of glycosaminoglycan (GAG) chains. The GAGs are linear co-polymers of N-acetyl-D-glycosamine [binding heparan sulfate] or N-acetyl-D-galactosamine [binding chondroitin sulfate (CS) chains] and aoidic sugars which are attached to these core proteins via a linking tetrasaccharide moiety. Three major classes of HS-carrying core proteins are present in living endothelial cells: cell membrane-spanning syndecans, GPI-linked glypicans, and a secreted perlecan core protein [Rosenberg et al., J. Clin. Invest. 99: 2062–2070 (1997)]. While the perlecan and glypican classes carry and bear HS chains almost exclusively, the syndecan core proteins are capable of carrying both HS and CS chains extracellularly. The appearance of specific glycosaminoglycan chains (such as HS and/or CS) extracellularly on protein cores is regulated both by the structure of the particular core protein as well as via the function of the Golgi apparatus intracellularly in a cell-type specific manner [Shworak et al., J. Biol. Chem. 269: 21204–21214 (1994)].
The syndecan class is composed of four closely related family proteins (syndecan-1, -2, -3 and -4 respectively) coded for by four different genes in-vivo. Syndecans-1 and -4 are the most widely studied members of this class and show expression in a variety of different cell types including epithelial, endothelial, and vascular smooth muscle cells, although expression in quiescent tissues is at a fairly low level [Bernfield et al., Annu. Rev. Cell Biol. 8: 365–393 (1992); Kim et al., Mol. Biol. Cell 5: 797–805 (1994)]. Syndecan-2 (also known as fibroglycan) is expressed at high levels in cultured lung and skin fibroblasts, although immunocytochemically this core protein is barely detectable in most adult tissues. However, syndecan-3 (also known as N-syndecan) demonstrates a much more limited pattern of expression, being largely restricted to peripheral nerves and central nervous system tissues (although high levels of expression are shown in the neonatal heart) [Carey et al., J. Cell Biol. 117: 191–201 (1992)]. All members of the syndecan class are capable of carrying both HS and CS chains extracellularly, although most of syndecan-associated biological effects (including regulation of blood coagulation, cell adhesion, and signal transduction) are largely thought to be due to the presence of HS chains capable of binding growth factors, or cell adhesion receptors and other biologically active molecules [Rosenberg et al., J. Clin. Invest. 99: 2062–2070 (1997)].
Curiously, however, very little is presently known about and relatively little research attention has been paid to the function of the syndecan core proteins in-situ. Syndecan-1 expression has been observed during development suggesting a potential role in the epithelial organization of the embryonic ectoderm and in differential axial patterning of the embryonic mesoderm, as well as in cell differentiation [Sutherland et al., Development 113: 339–351 (1991); Trautman et al., Development 111: 213–220 (1991)]. Also, mesenchymal cell growth during tooth organogenesis is associated with transient induction of syndecan-1 gene expression [Vainio et al., Dev. Biol. 147: 322–333 (1991)]. Furthermore, in adult living tissues, expression of syndecan-1 and syndecan-4 proteoglycans increases within arterial smooth muscle cells after balloon catheter injury [Nikkari et al., Am. J. Pathol. 144: 1348–1356 (1994)]; in healing skin wounds [Gallo et al., Proc. Natl. Acad. Sci. USA 91: 11035–11039 (1994)]; and in the heart following myocardial infarction [Li et al., Circ. Res. 81: 785–796 (1997)]. In the latter instances, the presence of blood-derived macrophages appears necessary for the induction of syndecan-1 and -4 gene expression. However, the effects of changes in syndecan expression on cell behavior are presently not well understood. For example, this core protein is believed to mediate bFGF binding and cell activity. Overexpression of syndecan-1 in 3T3 cells led to inhibition of bFGF-induced growth [Mali et al., J. Biol. Chem. 268: 24215–24222 (1993)]; while in 293T cells, overexpression of syndecan-1 augmented serum-dependent growth [Numa et al., Cancer Res. 55: 4676–4680 (1995)]. Furthermore, syndecan-1 overexpression showed increased inter-cellular adhesion in lymphoid cells [Lebakken et al., J. Cell Biol. 132: 1209–1221 (1996)] while also decreasing the ability of B-lymphocytes to invade collagen gels [Libersbach, B. F. and R. D. Sanderson, J. Biol. Chem. 269: 20013–20019 (1994)]. These ostensibly contradictory findings have merely added to the uncertainty and the disparity of knowledge regarding the effects of syndecan expression.
In comparison, the glypican core protein class is composed of five murine and human members and a Drosophila dally homologue [Rosenberg et al., J. Clin. Invest. 99: 2062–2070 (1997)]. Unlike syndecans, the glypican members are fully extracellular proteins attached to the cell membrane via a GPI anchor. Only one member of the class, glypican-1, is expressed in endothelial cells. Another unique feature of the glypican class of proteoglycans is that they carry substantially only heparan sulfate (HS) chains [Aviezer et al., J. Biol. Chem. 269: 114–121 (1994)]. Consequently, while little is presently known about the biological function of glypicans, they appear able to stimulate FGF receptor 1 occupancy by bFGF and appear able to promote biological activity for several different FGF family members [Steinfeld et al., J. Cell Biol. 133: 405–416 (1996)].
Finally, perlecan is the third and last class of heparan sulfate (HS)-carrying core proteins. Perlecan is a secreted proteoglycan that also has been implicated in regulation of bFGF activity [Aviezer et al., Mol. Cell Biol. 17: 1938–1946 (1997); Steinfeld et al., J. Cell Biol. 133: 405–416 (1996)]. However, little is known regarding this basal lamina proteoglycan beyond its interaction with basic fibroblast growth factor receptor.
In sum therefore, it is evident that the quantity and quality of knowledge presently available regarding glycoseaminoglycan (GAG) binding core proteins is factually incomplete, often presumptive, and in some instance apparently contradictory. Clearly the rule of specific proteoglycans as mediators under varying conditions is recognized; nevertheless, the mechanisms of action and the functional activity of the various individual classes of core proteins yet remains to be elucidated in full. Thus, while the role of proteoglycans in some manner relates to angiogenesis, there is no evidence or data known to date which clearly establishes the true functional value of proteoglycans nor which establishes a use for proteoglycans as a means for stimulating angiogenesis in-situ.